DIMERIZATION ASSAY

Abstract

Disclosed are methods, kits and cells for screening an inhibitor of association between candidate binding partners, such as for screening antagonists of amyloid peptides. The methods, kits and cells employ a reporter expression cassette and hybrid proteins. The reporter expression cassette encodes a reporter and comprises at least one DNA binding site. Each hybrid protein comprises a candidate binding partner and a component of a DNA binding protein and, upon association, form a DNA-binding complex capable of binding to the at least one binding site and inhibiting expression of the reporter. The methods, kits and cells find application, for example, in the identification of inhibitors that may be useful in treating diseases associated with protein aggregation, such as Alzheimer's Disease and Parkinson's Disease.

Claims

1. A method for screening for an inhibitor of association between first and second candidate binding partners, the method comprising: providing a cell, wherein the cell comprises: a test compound; a first hybrid protein comprising a first component of a DNA-binding protein linked to the first candidate binding partner; a second hybrid protein comprising a second component of the DNA-binding protein linked to the second candidate binding partner; and a reporter expression cassette that encodes a reporter expression product, wherein the first and second hybrid proteins form a DNA-binding complex upon association of the first and second candidate binding partners, and wherein the reporter expression cassette comprises at least one binding site for the DNA-binding complex such that binding of the complex to the binding site inhibits expression of the reporter expression product; and determining expression of the reporter expression product in the presence of the test compound; wherein an increase in expression of the reporter expression product in the presence of the test compound indicates that the test compound is capable of inhibiting association between the first and second candidate binding partners.

2. The method of claim 1, wherein the reporter expression product is a reporter protein, optionally wherein the reporter protein is a cell survival protein, a cell reproduction protein, a fluorescent protein, a bioluminescent protein, a protease, an enzyme that acts on a substrate to produce a colorimetric signal, a protein kinase, a transcriptional activator, or a regulatory protein such as ubiquitin.

3. The method of claim 2, wherein the reporter protein is a cell survival protein, optionally wherein the cell survival protein is an enzyme involved in synthesising compounds that are required for cell survival, or a protein that is able to inhibit action of a toxic agent.

4. The method of claim 3, wherein the cell survival protein is an exogenous cell survival protein that is able to compensate for a deficiency in an endogenous cell survival protein; and wherein the method is performed under selection conditions such that survival of the cell is dependent upon activity of the exogenous cell survival protein.

5. The method of claim 4, wherein the cell survival protein is dihydrofolate reductase (DHFR), optionally wherein the DHFR has an amino acid sequence that is at least 80% identical to the sequence set forth in SEQ ID NO: 1.

6. The method of claim 1, wherein the reporter expression cassette comprises between 1 and 5, between 1 and 10, between 1 and 15, between 1 and 20, between 5 and 10, between 5 and 15, between 5 and 20, between 10 and 15, between 10 and 20, between 10 and 18 or between 12 and 16 binding sites.

7. The method of claim 2, wherein some or all of the binding site(s) are located in the protein coding sequence of the reporter expression cassette.

8. The method of claim 1, wherein the first and second components of the DNA-binding protein have an identical amino acid sequence.

9. The method of claim 1, wherein the first and second components of the DNA-binding protein are DNA-binding fragments of a eukaryotic transcription factor, optionally a human transcription factor.

10. The method of claim 9, wherein the first and second components of the DNA-binding protein are DNA-binding fragments of a basic leucine zipper (bZIP), basic helix-loop helix (bHLH) or bHLH leucine zipper (bHLH-Zip) transcription factor, and optionally wherein a) the at least one binding site is a TPA response element (TRE) having the nucleotide sequence TGACTCA (SEQ ID NO: 5) or TGAGTCA (SEQ ID NO: 6); b) the at least one binding site is an Ebox response element having the nucleotide sequence CACGTG (SEQ ID NO: 7) or CACATG (SEQ ID NO: 8); c) the at least one binding site is a CCAAT binding site having the nucleotide sequence ATTGCGCAAT (SEQ ID NO: 9); d) the at least one binding site is a cAMP response element (CRE) having the nucleotide sequence TGACGTCA (SEQ ID NO: 10); e) the at least one binding site is a Maf recognition element (MARE) having the nucleotide sequence TGCTGA.sup.G/.sub.CTCAGCA (SEQ ID NO: 32) or TGCTGA.sup.GC/.sub.CGTCAGCA (SEQ ID NO: 33); or f) the at least one binding site is a PAP/CREB-2/PAR binding site having the nucleotide sequence TTACGTAA (SEQ ID NO: 34).

11. The method of claim 1, wherein the first and second candidate binding partners are capable of forming protein aggregates, optionally wherein the first and second candidate binding partners are amyloid peptides.

12. The method of claim 11, wherein a) the first and second candidate binding partners are amyloid-β (Aβ) peptides, optionally wherein the Aβ peptides comprise an amino acid sequence having the sequence of SEQ ID NO: 49; or b) the first and second candidate binding partners are α-synuclein (αS) polypeptides, optionally wherein the αS polypeptides comprise an amino acid sequence having the sequence of SEQ ID NO: 53.

13. A fusion protein comprising a component of a DNA-binding protein and an amyloid peptide component capable of dimerization; wherein said fusion protein forms a complex capable of binding DNA upon dimerization via the amyloid peptide component.

14. The fusion protein of claim 13, wherein the amyloid peptide component is: a) an amyloid-β (Aβ) peptide, optionally wherein the Aβ peptide has the amino acid sequence set forth in SEQ ID NO: 49; or b) an α-synuclein (αS) polypeptide, optionally wherein the αS polypeptide has the amino acid sequence set forth in SEQ ID NO: 53.

15. The fusion protein of claim 14, wherein the DNA-binding component comprises an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 47, optionally wherein the fusion protein comprises an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 51 or 55.

16. A fusion protein expression cassette encoding the fusion protein of claim 13.

17. A kit comprising: a reporter expression cassette that encodes a reporter expression product; and one or more fusion protein expression cassettes encoding a first and second fusion protein; wherein the first fusion protein comprises a first component of a DNA-binding protein and a first candidate binding partner, wherein the second fusion protein comprises a second component of a DNA-binding protein and a second candidate binding partner, wherein the first and second fusion proteins form a DNA-binding complex upon association of the first and second candidate binding partners; and wherein the reporter expression cassette comprises at least one binding site for the DNA-binding complex such that binding of the DNA-binding complex to the binding site inhibits expression of the expression product.

18. (canceled)

Description

SUMMARY OF THE FIGURES

[0269] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0270] FIG. 1. General principles of the Transcription-Block Survival (TBS) Assay.

[0271] Fifteen TREs have been introduced into the mDHFR gene. A) The changes to the DNA sequence result in a fully active protein that expresses, folds, and confers survival in M9 minimal media using trimethoprim (TMP) to inhibit bacterial DHFR. B) Basic-cJun can form DNA-bound homodimers and its expression prevents mDHFR transcription and no colonies grow. M9 agar plates with trimethoprim in the absence of IPTG (left hand side plates) do not form colonies. Expressing mDHFR with IPTG (right hand side plates), generates colonies in A) but not B).

[0272] FIG. 2. Schematic of the amyloid-TBS assay for detecting inhibitors of αS dimerization

[0273] This intracellular assay utilises BL21 E. coli expressing basic-αS and a reporter plasmid, mDHFR. DHFR is an essential protein and under the specific inhibition of the endogenous bacterial form using the antibiotic Trimethoprim (Tmp), the transcription and subsequent expression of mDHFR is essential for cell survival. BL21 E. coli are co-transformed with 2 plasmids. Firstly, the mDHFR plasmid which contains silent mutations that preserve the native structure and function of the expressed mDHFR protein but provides the specific 2-O-tetradecanoylphorbol-13-acetate response element (TRE) DNA-binding motifs for the basic regions of c-Jun to bind. Secondly, Basic-αS, encoding recombinant αS with the DNA-binding basic regions of c-Jun attached to the N-terminus. On aggregation of basic-αS, the basic regions come together and bind the TRE binding sites in the mDHFR reporter plasmid. This prevents DNA polymerase progression and halts mDHFR transcription, causing cell death. As a cell death signal confers primary events of the αS misfolding cascade and aggregation, successful inhibitors of these events will allow mDHFR transcription and can be identified via cell survival and growth of colonies. Basic regions adapted from PDB 1 a02 using Swiss-PdbViewer (Version 4.1.0). CmR, chloramphenicol resistance; AmpR, ampicillin resistance.

[0274] FIG. 3. The amyloid-TBS assay can be used to identify compounds that bind and sequester αS peptides as monomers.

[0275] A) Fifteen TREs have been introduced into the mDHFR gene and this results in a fully active protein as described in FIG. 1A. B) Basic-αS can form DNA-bound homodimers and its expression prevents mDHFR transcription and no colonies grow. C) Peptides that bind to the aggregate form of αS but that fail to target the monomer result in paired DNA-binding domains which therefore do not dissociate the DNA-bound complex. This will not rescue transcription of the mDHFR gene and no colonies are observed. D) Inhibitor expression results in the basic-αS complex dissociating from TRE sites on the mDHFR gene leading to the restoration of mDHFR transcription-translation and colony formation. Consistent with this result, M9 agar plates with trimethoprim in the absence of IPTG (left hand side plates) do not form colonies. Expressing mDHFR with IPTG (right hand side plates), generates colonies in A) and D) but not B) or C).

[0276] FIG. 4. Schematic of the Aβ.sub.1-42 dimerization assay

[0277] Schematic illustrating general principles of using the TBS assay to screen for inhibitors of the initial dimerization event of Aβ.sub.1-42. In bacterial cells, endogenous DHFR can be inhibited using the antibiotic trimethoprim, making cells reliant on the modified, exogenous DHFR (top panel). In this setting, if AP-1 binds to the TRE sites in the DHFR, transcription is blocked and no functional DHFR enzyme is produced, resulting in cell death (middle panel). By attaching this basic cJun region to Aβ.sub.1-42, a functional DNA binder will be created if two Aβ.sub.1-42 peptides dimerize. In the absence of an inhibitor, basic-Aβ.sub.1-42 dimerization would lead to blocking of the DHFR gene transcription, resulting in cell death (lower left hand panel). Only cells treated with a successful Aβ.sub.1-42 dimerization inhibitor would produce DHFR and so survive, allowing selection for potential therapeutics for treating Alzheimer's Disease (lower right hand panel).

EXAMPLES

Example 1—Development of a Generalised Approach to Identify Inhibitors of Dimerization

[0278] Many rational design approaches, randomised screening approaches, and selection systems result in the successful identification of compounds capable of binding to given protein targets. However, what is much more difficult to ensure, is that binding to said target will result in ablating target protein function. There are many instances where formation of a protein-protein interaction (PPI) has not ensured loss of function. To address this major bottleneck in antagonist screening and design, we have taken inspiration from the transcription factor DNA-binding system and reversed their role in transcription.

[0279] Introducing DNA-Binding Sites into the DHFR Gene

[0280] It can be difficult to predict whether a compound that is derived to bind to given protein target will antagonise its function. To tackle this we have taken the gene corresponding to the essential enzyme, dihydrofolate reductase (DHFR), and introduced 15 TPA response elements (TREs) into the gene. This has been achieved using a combination of both silent and conserved mutations, such that the activity of the enzyme is preserved.

[0281] All changes have been made in solvent exposed regions of the molecule to minimise the structural perturbations, with several proposed changes removed via close inspection of the accessible surface area (ASA) within the pdb file (PDBid=2FZJ (Cody et al. (2006)). This was done by inputting the pdb file into the ASA calculator at http://cib.cf.ocha.ac.jp/bitool/ASA/. A cut-off value of 20 was used—residues that had an ASA value lower than this were considered to be buried and not modified; residues that had an ASA value greater than this are considered exposed.

[0282] No changes have been made in residues deemed important for catalysis or NADPH binding. Methods of identifying the solvent exposed regions of the reporter protein are known. For example, it is possible to take the coordinate files for the reporter protein, e.g. a protein databank (PDB) file and use a program that calculates the accessible surface area (ASA) which informs the user how exposed/buried residues are within a structure. An exemplary ASA program can be found at http://cib.cf.ocha.ac.jp/bitool/ASA/. An exemplary cut-off value of 20 can be used, such that residues that are lower than this are considered to be buried and greater than this are considered exposed. In this way, the locations of solvent exposed residues can be identified and codons modified accordingly.

[0283] Shown below is the sequence of the mDHFR gene (SEQ ID NO: 11) with DNA mutations bold and underlined and changes within the translated protein sequence (SEQ ID NO: 31) shown. Shown in bold italics are the NheI and HindIII sites used for subcloning the gene into the pES300d vector. Mutations were made by inspection of the desired consensus sequences (TGACTCA or TGAGTCA) and all three frames and the corresponding changes to the amino acid sequence upon making the necessary single base-pair changes. For example, either of the two desired sequences above can be put into any one of the three reading frames and the corresponding amino acid sequence and tolerated variations can be given:

TABLE-US-00008 i) Frame 1: TGA CTC Axx 1 = stop 2 = LV 3 = I/M/T/N/K/S/R ii) Frame 2: xTG ACT CAx 1 = LMV 2 = TS 3 = HQ iii) Frame 3: xxT GAC TCA 1 = FSYCLPHRITNVADG 2 = DE 3 = S

[0284] This gives rise to a number of codons to be identified for silent mutation and consequently a number of options for conserved or semi-conserved mutations that would permit the introduction of TREs into the mDHFR gene: [0285] i) No options [0286] ii) LSH, LSQ, LTH, LTQ, MSH, MSQ, MTH, MTQ, VSH, VSQ, VTH, VTQ [0287] iii) ADS, AES, CDS, CES, DDS, DES, FDS, FES, GDS, GES, HDS, HES, IDS, IES, LDS, LES, NDS, NES, PDS, PES, RDS, RES, SDS, SES, TDS, TES, VDS, VES, YDS, YES

[0288] From this we were able to implement the following changes into the mDHFR gene to give minimum perturbation to the overall sequence. Where possible mutations were silent or conservative. All mutations were also placed at solvent exposed sites and away from the catalytic centre (E116) and away from residues required for NADPH/substrate binding (A10/R71). This resulted in the introduction of 15 TREs into the mDHFR gene:

TABLE-US-00009 1. VSQ (silent) = GTG AGT CAG 2. NEF.fwdarw.NES (F32S) = AAT GAG TCA 3. MTT.fwdarw.MTQ (T40Q) = ATG ACT CAG 4. TSS.fwdarw.TDS (S42D) = ACT GAC TCA 5. VEG.fwdarw.VES (G46S) = GTT GAG TCA 6. PEK.fwdarw.PES (K64S) = CCT GAG TCA 7. LSR.fwdarw.LSQ (R78Q) = CTG AGT CAA 8. IEQ.fwdarw.IES (Q103S) = ATT GAG TCA 9. VDM.fwdarw.VDS (M112S) = GTT GAC TCA 10. MNQ.fwdarw.MTQ (N127T) = ATG ACT CAA 11. VTR.fwdarw.VTQ (R138Q) = GTG ACT CAG 12. FES (silent) = TTT GAG TCA 13. IDL.fwdarw.IDS (L154S) = ATT GAC TCA 14. PEY.fwdarw.PES (Y163S) = CCT GAG TCA 15. LSE.fwdarw.LSQ (E169Q) = CTG AGT CAG

[0289] This design process gave rise to the following sequence:

TABLE-US-00010 A   S   V   R   P   L   N   C   I   V   A   V   S   Q   N   M   G  GTT CGA CCA TTG AAC TGC ATC GTC GCC   AAT ATG GGG I   G   K   N   G   D   L   P   W   P   P   L   R   N   E   S   K ATT GGC AAG AAC GGA GAC CTA CCC TGG CCT CCG CTC AGG   AAG Y   F   Q   R   M   T   Q   T   D   S   V   E   S   K   Q   N   L TAC TTC CAA AGA       AAA CAG AAT CTG V   I   M   G   R   K   T   W   F   S   I   P   E   S   N   R   P GTG ATT ATG GGT AGG AAA ACC TGG TTC TCC ATT   AAT CGA CCT L   K   D   R   I   N   I   V   L   S   Q   E   L   K   E   P   P TTA AAG GAC AGA ATT AAT ATA GTT   GAA CTC AAA GAA CCA CCA R   G   A   H   F   L   A   K   S   L   D   D   A   L   R   L   I CGA GGA GCT CAT TTT CTT GCC AAA AGT TTG GAT GAT GCC TTA AGA CTT  E   S   P   E   L   A   S  K    V   D   S   V   W   I   V   G   G  CCG GAA TTG GCG AGC AAA   GTT TGG ATC GTC GGA GGC S   S   V   Y   Q   E   A   M   T   Q   P   G   H   L   R   L   F AGT TCT GTT TAC CAG GAA GCC   CCA GGC CAC CTT AGA CTC TTT V   T   Q   I   M   Q   E   F   E   S   D   T   F   F   P   E   I  ATC ATG CAG GAA   GAC ACG TTT TTC CCA GAA  D   S   G   K   Y   K   L   L   P   E   S   P   G   V   L   S   Q  GGG AAA TAT AAA CTT CTC   CCA GGC GTC  V   Q   E   E   K   G   I   K   Y   K   F   E   V   Y   E   K   K GTC CAG GAG GAA AAA GGC ATC AAG TAT AAG TTT GAA GTC TAC GAG AAG AAA D   *   A   * GAC T AA

[0290] We have introduced 15 TREs via silent and conserved mutations into solvent exposed positions within the gene coding for the essential enzyme dihydofolate reductase (DHFR). We demonstrate that these changes result in a functional enzyme. Under selective conditions introduction of AP-1 prevents DHFR expression by binding to TRE sites within the gene, blocking transcription, and preventing colony formation under selective conditions (FIG. 1A). In contrast, attenuated versions of AP-1 that lack a basic DNA-binding region fail to prevent colony formation.

[0291] Testing Functionality of DHFR Protein

[0292] The selection system is based on the fact that bacterial DHFR can be specifically inhibited using trimethoprim, rendering cells dependent upon murine DHFR (mDHFR) activity for their survival. The first test of the system was to establish that mDHFR protein refolds and is active. SDS-PAGE analysis was used to confirm that the protein is highly expressed upon addition of IPTG. Further evidence that the protein is expressed, folds, and is functionally active was verified by transformation of bacterial cells and confirmed by the presence of multiple colonies in minimal media containing trimethoprim.

[0293] Establishing an Assay that Uses Cell Survival as a Readout of DNA-Binding Activity

[0294] It was next necessary to establish that introduction of an AP-1 component (in this case basic-cJun) would result in binding to the 15 TRE's introduced within the mDHFR gene and therefore failure of the gene to be transcribed.

[0295] Three plasmids were used for this assay. These are i) p300-mDHFR (Cm; SEQ ID NO: 42) to express the 12×consensus sequence containing mDHFR, which is under control of the lac-operon; ii) p230d-basic-cJun (Amp; SEQ ID NO: 43) which is also under control of the lac-operon; iii) pREP4 (Kan; SEQ ID NO: 44) to express the lac repressor.

[0296] Cells were grown under non-selective conditions (i.e. LB/LB agar) containing Cm/Amp/Kan up until the time of the Assay. During TBS Selection Cells are grown in M9 minimal media (Agar or Broth) in the presence of Cm/Amp/Kan, as well as Tmp (to inhibit the bacterial copies of DHFR) and IPTG (to induce expression of mDHFR and bZIP proteins). During Assay selection, media-lacking ITPG is used to serve as a negative control to ensure that cell survival is exclusively driven by the loss on interaction between bZIP target protein and the consensus sequences located within the mDHFR gene.

[0297] As expected, overexpression of basic-cJun on the second plasmid resulted in a complete loss of bacterial colonies in minimal media (FIG. 1B). Without wishing to be bound by theory, it is believe that this works because AP-1 binds to the multiple TREs found within the mDHFR gene and therefore works in the opposite way to its natural function. Rather it works by blocking transcription and preventing the machinery from moving along the DNA. As a control a version of cJun containing the leucine zipper, but lacking in the DNA-binding basic region (SEQ ID NO: 45), was tested. As expected this version did not prevent bacterial colony formation in minimal media.

[0298] Discussion

[0299] We have shown using the essential enzyme mDHFR that i) enzymatic activity is preserved upon introduction of 15 TREs into the gene under selective conditions activity becomes lost when basic-cJun is introduced, and the basic region within basic-cJun is an absolute requirement for this loss of mDHFR activity. This assay therefore uses cell survival as a marker to allow rapid screening of peptide libraries.

Example 2—Designing a Cell Assay to Detect Inhibitors to Primary Events in α-Synuclein Aggregation

[0300] The assay described in Example 1 demonstrates that an engineered mDHFR gene can be used to detect DNA binding of AP-1 to DNA-binding sites located in the gene and allows for the selection of cells that contain unbound DHFR. It was then proposed to develop a cell-based assay using this mechanism to screen for inhibitors of primary events in α-Synuclein (αS) aggregation.

[0301] Generating Components of the Assay

[0302] To establish the assay, the DNA-binding basic region of the AP-1 (Activator-protein 1) receptor subunit c-Jun was attached to the N-terminus of αS. AP-1 is a dimeric transcription factor which assembles via a bZIP domain comprising a DNA-binding basic region and a leucine zipper (Seldeen et al. 2009). The AP-1 receptor can comprise a homodimer of Jun proteins or a Jun-Fos heterodimer (Nakabeppu et al. 1988; Sassone-Corsi et al. 1988) and consequently the basic regions of c-Jun were chosen for Basic-αS. The basic region contains positively charged residues for the interaction with TRE sites in DNA containing the conserved motif 5′-TGA G/C TCA-3′. For the assay, the mDHFR reporter plasmid described in Example 1 was used, which contains silent and conserved mutations giving a total of 15 TRE sites.

[0303] Firstly, basic-αS DNA was successfully synthesised via PCR. Following the successful amplification and purification of basic-αS, the DNA insert was subcloned into a p230d plasmid. This plasmid was chosen due to its complementarity with the subsequent DHFR(TRE)-p300d plasmid to be used for the assay, both expressing distinct antibiotic resistance. Sequencing confirmed the generation of the p230-basic-αS plasmid.

[0304] A pREP4 plasmid was used to encode the lacI gene for regulating expression of mDHFR under the control of the lac operon. IPTG is used to remove the lacI repressor and induce transcription of mDHFR. Under minimal media conditions in which endogenous bacterial DHFR is inhibited, the transcription of mDHFR is essential for cell survival. Therefore, during testing of the assay, negative controls on minimal media lacking IPTG were expected to grow no colonies. Under test conditions with IPTG, mDHFR transcription would yield colonies unless inhibited by other variables.

[0305] BL21 E. coli cells harbouring the pREP4 plasmid were co-transformed with DHFR(TRE)-300d and either αSp230d or Basic-αS p230d. Cells were load-matched and plated under three conditions with the overexpression of αS and Basic-αS being the stimulation to aggregate. Positive controls grown on LB agar under selection shows the cells were live and contained all 3 plasmids. M9 minimal agar contained Tmp antibiotic to specifically inhibit the endogenous DHFR protein. Test plates contained IPTG for expression of mDHFR under control of the lac operon. Negative control plates were expected to not produce colonies in the absence of IPTG as pREP4, expressing the lacI repressor, prevents transcription of mDHFR.

[0306] Testing the Amyloid-TBS Assay

[0307] As described above in Example 1, introducing 15 TRE's into the mDHFR gene via silent and conserved mutations resulted in a functional DHFR enzyme, which can be used to maintain cell growth under selective conditions (FIG. 2A). Co-expression of basic-αS leads to occupation of TRE sites on the DHFR gene and blocks transcription of the engineered mDHFR, resulting in cell death (FIG. 2B). As a final proof that this approach is successful, when a peptide (45-54W) designed to bind to αS was co-expressed with the engineered mDHFR and basic-αS cell survival is favoured by the loss of basic-αS DNA binding activity (FIG. 2D). Cell survival was not observed when a control peptide (a scrambled dummy sequence that does not bind αS) was co-expressed with the engineered mDHFR and basic-α (FIG. 2C).

[0308] Discussion

[0309] αS aggregation forms large, ordered fibrils which are found in Lewy Body inclusions of dopaminergic neurons in patients with Parkinson's Disease (PD). Despite the exact underlying cause for PD being unclear, one strategy for treatment is to prevent αS aggregation. As the disordered nature of αS prevents traditional drug design, this study aimed to develop a new assay for the intracellular screening of αS aggregation inhibitors (FIG. 2). In particular, this assay allows for the selection of inhibitor that block initial dimer formation of αS.

[0310] We synthesised basic-αS, which comprises the DNA-binding basic region of c-Jun attached to αS. Normally, c-Jun bind DNA as pairs, with dimerization facilitated by a coiled-coil dimerization domain. However, in the amyloid-TBS assay, dimerization is instead achieved by the αS domains appended to the basic peptide (i.e. no coiled-coil is present in basic-αS). Aggregation of αS causes the basic regions to come in close proximity, as they would in AP-1, to be able to bind TRE sites in a reporter mDHFR plasmid. This prevents mDHFR transcription such that primary events in the aggregation of basic-αS could be detected as a cell death signal when compared to WT αS (FIG. 1B).

[0311] In order to establish that the assay could be used to identify inhibitors of the initial αS dimerization event, we made use of 45-54W, a peptide inhibitor that had previously been evaluated as being able to bind αS and reduce aggregation levels at early stages of the misfolding pathway (Cheruvara et al. 2015). It was not conclusive from previous studies whether 45-54W targeted the initial dimerization event. Use of 45-54W restored mDHFR transcription-translation and colony formation, indicating that the inhibitor binds and inhibits initial αS dimerization (FIG. 1D). Importantly, the restoration of colony formation was not observed when a control peptide was used that binds αS but not in the monomeric form (FIG. 1C). Distinguishing between binding and inhibition of dimerization is important since αS binders derived by other means do not necessarily prevent dimer formation (and the subsequent formation of higher-n oligomers, and their conformers). Therefore, such compounds may not translate into functional antagonists of αS pathology.

[0312] The TBS-based assay described here allows rapid screening of genetically encoded peptide libraries, to assess cell survival to consequently derive functionally active antagonists of αS pathology. Since peptide libraries are screened entirely inside living cells, the assay described here has the added benefit of removing library members that are toxic, susceptible to proteases, insoluble, or non-specific for αS and detrimental to cell growth. This assay can therefore advantageously be used to select for inhibitors that bind αS, inhibit dimerization and lack cell toxicity in a single step. Furthermore, the assay described here has the significant advantage of concomitantly interrogating exogenously applied peptides for membrane permeability (e.g. naturally, via strand-inducing constraints or CPP appendage), protease resistance, and lack of cytotoxicity.

[0313] Following identification of novel inhibitors using the amyloid-TBS assay, biophysical, structural and primary neuron-based cell biology approaches can be used to validate the inhibitor function. For example, the following assays can be used: i) continuous growth ThT experiments, demonstrate inhibition of amyloid formation in a dose-dependent manner ii) single molecule fluorescence and atomic force microscopy (AFM) imaging, confirm prevention of amyloid formation iii) circular dichroism spectroscopy experiments to detect changes in global secondary structure iv)neuronal cell assays to demonstrate reduced αS cytotoxicity v) intracellular delivery of peptides to test colocalization and downstream effects of the in cellulo derived peptides on cytotoxicity and proteostasis in neurons where wild-type or mutant αS is overexpressed. Undertaking these assays using our iterative strategy of Truncation, Randomisation and Selection (TraSe; Crooks et al. 2011) will lead to reduced size antagonists by identifying the smallest functional unit required for effective target binding.

[0314] In conclusion, this study shows an intracellular method to identify inhibitors that directly prevent αS aggregation at the initial step in the misfolding pathway. Aggregation of αS underlies the related synucleinopathies, in addition to PD, which means a successful disease-modifying lead could have broad benefits. Finally, with amyloid fibrils underlying other neurodegenerative diseases, the amyloid-TBS assay has the potential to be adapted to study inhibitors of other toxic protein aggregates.

Example 3—Designing a Cell Assay to Detect Inhibitors of Amyloid β1-42 Dimerization

[0315] The strategy described above was also used to design an assay to screen for inhibitors of primary events in Aβ.sub.1-42 dimerization.

[0316] By attaching this basic cJun region to Aβ.sub.1-42, a functional DNA binder will be created if two constructs dimerize. If introduced to bacteria cells reliant on modified DHFR, basic-Aβ.sub.1-42 dimerization would lead to blocking of the DHFR gene transcription and so cell death. This provides the basis of a novel cell assay that could be used to find peptide inhibitors, as summarised in the schematic in FIG. 3. Only cells treated with a successful Aβ.sub.1_42 dimerization inhibitor would produce DHFR and survive, allowing selection for potential AD therapeutics.

[0317] Generating a Basic cJun-Aβ.sub.1-42 (Basic-Aβ.sub.1-42) Fusion Protein

[0318] The DNA binding moiety from cJun is a 25 amino acid coiled coil, made primarily of basic amino acids lysine, arginine and histidine (SEQ ID NO: 47). PCR was used to attach the DNA encoding this basic region (SEQ ID NO: 46) to Aβ.sub.1-42 DNA (SEQ ID NO: 48) followed by subcloning into a p230d vector. Sanger sequence was performed to confirm that the production of a p230d plasmid containing the basic-Aβ.sub.1-42 sequence (SEQ ID NO: 50).

[0319] Testing the Aβ Dimerization Assay

[0320] BL21 GOLD cells stably expressing a pREP4 plasmid containing lac inhibitor (lad) gene were used for the dimerization assay, so that the lac operon could be controlled. Cells were transformed with p300d plasmid containing DHFR with 15 TRE sites under control of the lac operon (described in Example 1), and either a p230d containing Aβ.sub.1-42 or constructed basic-Aβ.sub.1-42.

[0321] BL21 from the 100 μL plates were scraped into LB and selecting antibiotics and grown to an OD.sub.600 of 0.5. 100 μL of culture was plated onto positive control, negative control and test plates, as described in Table 3 below.

TABLE-US-00011 TABLE 3 Composition of positive, negative and test plates used in the Aβ.sub.1-42 dimerization assay Plate type Positive control Negative control Test Media LB agar M9 minimal M9 minimal agar agar Selecting 100 μM Cm, 100 μM Cm, 100 μM Cm, Antibiotics Kan and Amp Kan and Amp Kan and Amp Treatments — 3.4 μM Tmp 3.4 μM Tmp (1 mg/mL stock (1 mg/mL in DMSO) stock in DMSO) 1 mM IPTG

[0322] Results following a 48-hour incubation are shown in Table 4 below. A covering of cells was seen on both positive control plates, showing that BL21 cells were not affected by scraping and re-plating. Tmp was used to inhibit the endogenous, E. coli DHFR enzyme. Both basic-Aβ.sub.1-42 and Aβ.sub.1-42 expressing cells were shown to be reliant on the modified DHFR because limited growth was seen on M9 minimal media plates, consistent with the lac repressor protein from pREF4 preventing expression of modified DHFR and resulting in a lack of THFA. Two basic-Aβ.sub.1-42 expressing colonies and one Aβ.sub.1-42 colony grew on these negative control plates. Test plates contained IPTG to bind to lac repressor protein and allow expression of the modified DHFR in the bacteria. There were 63 Aβ.sub.1-42 expressing BL21 colonies formed on these plates, which confirmed that the modified DHFR gene allows cells to grow on minimal media. Importantly, only 2 basic-Aβ.sub.1-42 expressing colonies grew on test plates, which was consistent with levels of background growth seen on negative control plates. This indicates that basic-Aβ.sub.1-42 protein dimerized and blocked modified DHFR leading to cell death. These results support the use of these cells in a basic-Aβ.sub.1-42 dimerization assay with cells only surviving if inhibition is achieved.

TABLE-US-00012 TABLE 4 Colony growth in basic-Aβ1-42 dimerization assay No. of colonies (CFU) basic-Aβ.sub.1-42 Aβ.sub.1-42 expressing expressing Media type Positive control Covered Covered Negative control 1 2 Test 63 2

[0323] Discussion

[0324] Inhibition of Aβ.sub.1-42 dimerization prevents formation of all types of Aβ.sub.1-42 oligomers making it an attractive therapeutic approach for AD. To screen for peptide inhibitors of this event, an in-cell detection assay was proposed using E. coli BL21 GOLD cells reliant on a modified DHFR containing cJun binding sites. The DNA binding moiety from cJun was successfully cloned onto the N-terminus of Aβ.sub.1-42 such that when expressed in these BL21 cells, the dimerized protein blocked DHFR transcription and lead to cell death.

[0325] Cloned p230d-basic-Aβ.sub.1-42 DNA was used to create an A431-42 dimerization assay. BL21 GOLD cells containing pREF4 were used for their protein expression ability and for the lacI gene to control the lac operon. Cells were transformed with p300d encoding modified DHFR DNA, and either p230d-basic-A431-42 or p230d-Aβ.sub.1-42. Results in Table 4 showed that on M9 minimal media, the lac repressor from pREF4 prevented expression of modified DHFR and resulted in a lack of THFA and so cell death. Two basic-Aβ.sub.1-42 expressing colonies and one Aβ.sub.1-42 colony grew on these negative control plates, perhaps due to insufficient exposure to Tmp, and so a higher concentration than 3.5 μM could be used if repeated to ensure all endogenous DHFR was inhibited. In the presence of IPTG, modified DHFR was expressed in the bacteria allowing for survival of Aβ.sub.1-42 expressing BL21 colonies (Table 4). Only 2 basic-Aβ.sub.1-42 expressing colonies survived in these conditions, indicating basic-Aβ.sub.1-42 protein dimerized and blocked modified DHFR as expected. This gives confidence that basic-Aβ.sub.1-42 expression can be used as a screening assay, with cells surviving if inhibition is achieved.

[0326] This BL21 GOLD assay system benefits from a survival endpoint making it possible to easily screen large libraries of peptides for dimerization inhibitors. The library of peptides could perhaps be designed using the dimerization interface of Aβ itself as a starting point as it is a self-dimer. Future experiments could demonstrate proof of principle using known inhibitors of amyloidosis such as the beta sheet breaker iAβ5 (Adessi and Soto, 2002) in order to show the expected level of cell growth of a successful inhibitor.

[0327] Peptides that inhibit the dimerization of basic cJun might be found in the assay which would not be useful for AD drug discovery. To overcome this, follow-up biophysical experiments monitoring peptide binding to wild-type Aβ.sub.1-42 protein could be used. For example, Surface Plasma Resonance or Isothermal Calorimetry could be performed to detect protein binding of peptides. X-ray crystallography of Aβ.sub.1-42 crystals soaked in potential inhibitors would also provide information about the mechanism of action of the inhibitor. Because the assay is performed in bacteria and not a disease relevant cortical neuron cell line, inhibitors will also require further testing to ensure the same effect is seen when in neurons or in vivo AD models. However, the assay does detect dimerization of human Aβ.sub.1-42 and so is useful for initial screening to finding peptides that can disrupt this.

[0328] In conclusion, expression of basic-Aβ.sub.1-42 in bacteria reliant on modified DHFR has created a novel system that detects Aβ.sub.1-42 dimerization.

Example 4—Library Creation

[0329] Dimerization Assay—Genetically Encoded Library Construction:

[0330] As described above, three plasmids were used for the dimerization assays. These are i) p300-mDHFR (Cm) to express the mDHFR containing 15 AP-1 binding sites, which is under control of the lac-operon; ii) p230d-basic-cJun fusion protein (basic-Aβ.sub.1-42 or basic-αS; Amp) which is also under control of the lac-operon; iii) pREP4 (Kan) to express the lac repressor.

[0331] Genetically encoded libraries are created using overlap extension PCR, subcloned into the p410d vector (Tet) and plated out. Each colony then represents a member of the library. We typically collect 2-5× the library size in colony numbers to gain approx. 95% total coverage. The maximum library size screenable using the approach is 10.sup.7. Once the library is complete colonies are pooled and mini-preparation of DNA performed. Finally the plasmid library is transformed into cells containing p300/p230/pREP4. During single step selection cells are plated onto LB agar (to demonstrate successful transformation), M9 agar lacking IPTG (as a negative control where no bZIP or mDHFR is expressed) and finally onto M9 agar containing Cm/Amp/Kan/Tet/Tmp/IPTG to drive production of basic-cJun fusion protein/mDHFR/Library such that cell viability is only restored if a given library member can prevent the cJun target from interacting with the cognate sequences within the mDHFR gene. Surviving colonies can next be pooled, grown and serially diluted in liquid cultures under selective conditions (M9 minimal medium with 1 μg/ml trimethoprim). Fastest growth, and hence the highest affinity interacting partners dominated the pool. Library pools as well as colonies from individual clones were sequenced to verify the arrival at one sequence. To assess library quality we sequence pools and single clones to find approximately equal distributions of varied amino acids. Pooled colonies exceeded the library size 5-10 fold. Using more recent ligation methods (Topo/Gibson/Gateway) it may be possible to move into the dimerization assay directly from ligation, giving the significant advantage of being able to screen larger libraries (possibly up to 10.sup.10 or 10.sup.11), however processes will need to be put into place (e.g. next gen sequencing) to ensure that library size and quality is fully represented prior to transformation into the dimerization assay.

[0332] Another possibility is to use pET24a as an alternative to the pREP4 vector used to express the lac repressor. This would allow the expression of both the lac repressor and library/antagonist off a single plasmid, i.e. avoiding the need for another antibiotic.

[0333] Dimerization Assay—Extracellular Compound Addition:

[0334] For extracellular libraries, cells containing p300-mDHFR plasmid are grown in the presence of p230d-basic-cJun fusion protein and pREP4 plasmids under non-selective conditions (LB agar/media). Once ready for assay overnights can then be placed into each well of microtitre plates (96, 384, 1536) at A600=0.05 and compound libraries screened by direct addition to each well. Plates are incubated at 37° C. and with shaking and successful compounds identified by monitoring of the absorbance signal at 600 nm. This extracellular compound addition method has the advantage of allowing the user to move away from standard peptide libraries (e.g. one can profile for helix constrained peptides, peptidomimetics, non-natural amino acids etc., or even small molecule libraries) and importantly allows the user to profile for cell penetrance concomitantly with the ability to inhibit dimerization. Once again, all proteins are under control of a lac promoter, and expression was induced with Isopropyl β-D-1-thiogalactopyranoside (IPTG).

[0335] Selection of Winner Peptides

[0336] Briefly, during the assay peptides (intracellular) or compounds (extracellular) that can disrupt dimerization of the basic-cJun fusion proteins will result in colony formation/cell growth on M9 minimal medium plates/media with 1 μg/ml trimethoprim to inhibit bacterial DHFR.

Example 5—Introducing the CRE, CCAAT and Ebox Binding Sites into the DHFR Gene

[0337] Constructs were designed whereby the CRE, CCAAT and Ebox binding sites, respectively, were inserted into the DHFR gene. These constructs can be tested in the assays described above.

[0338] Inserting the CRE Binding Site into the DHFR Gene

[0339] CRE is usually defined as TGACGTCA (SEQ ID NO: 10). Mutations in the DHFR gene can be made by inspection of the desired consensus sequence and all three frames and the corresponding changes to the amino acid sequence upon making the necessary single base-pair changes. The CRE is 8 bp as so can span four codons. For example, the sequence defined above can be put into any one of the three reading frames and the corresponding amino acid sequence and tolerated variations can be given:

TABLE-US-00013 A: Frame 1: TGA CGT CAx 1 = stop 2:R 3:H/Q B: Frame 2: xTG ACG TCA 1:LMV 2:T 3:S C: Frame 3: xxT GAC GTC Axx 1:FLIVSPTAYHNDCRG 2:D 3:V 4:IMTNKSR

[0340] From this it is possible to implement changes into the mDHFR gene to give minimal perturbation to the overall sequence. Mutations should be placed at solvent exposed sites and away from the catalytic centre and where possible mutations should be silent or conservative.

[0341] An example of an mDHFR gene that is modified to contain CRE binding sites is shown as follows:

TABLE-US-00014 ATGGTTCGACCATTGAACTGCATCGTCGCCGTGTCCCAAAATATGGGGATTGGCAAGAACGGAGACCTAC CCTGGCCTCCGCTCAGGAACGAGTTCAAGTACTTCCAAAGAATGACGTCAACCTCTTCAGTGGAAGGTAA ACAGAATCTGGTGATTATGGGTAGGAAAACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGAC AGAATTAATATAGTGACGTCAAGAGAACTCAAAGAACCACCACGAGGAGCTCATTTTCTTGCCAAAAGTT TGGATGATGCCTTAAGACTTATTGAACAACCGGAATTGACGTCAAAAGTAGACATGGTTTGGATCGTCGG AGGCAGTTCTGTTTACCAGGAAGCCATGAATCAACCAGGCCACCTTAGACTCTTTGTGACGTCAATCATG CAGGAATTTGAAAGTGACACGTTTTTCCCAGAAATTGATTTGGGGAAATATAAACTTCTCCCAGAATACC CAGGCGTGACGTCAGAGGTCCAGGAGGAAAAAGGCATCAAGTATAAGTTTGAAGTCTACGAGAAGAAAGA CTAAGCTTAA

[0342] Nucleotide residues in bold underline indicate consensus CRE binding sites.

[0343] Nucleotide residues in lowercase and italics correspond to the restriction enzyme sites for AscI and HindIII at the 5′ and 3′ ends of the sequence, respectively.

[0344] The resulting amino acid sequence is shows as follows:

TABLE-US-00015 M V R P L N C I V A V S Q N M G I G K N G D L P W P P L R N E F K Y F Q R M T  T S S V E G K Q N L V I M G R K T W F S I P E K N R P L K D R I R I V   S R E L K E P P R G A H F L A K S L D D A L R L I E Q P E L   S K V D M V W I V G G S S V Y Q E A M N Q P G H L R L F V T   I M Q E F E S D T F F P E I D L G K Y K L L P E Y P G V   S E V Q E E K G I K E K F E V Y E K K D

[0345] Amino acid residues in italics are solvent exposed residues. The other residues are classed as buried residues.

[0346] Amino acid residues in bold underline are residues that have been altered as a result of the insertion of CRE into the nucleotide sequence.

[0347] A summary of the amino acid changes is provided as follows:

TABLE-US-00016 1. MTT.fwdarw.MTS (T40S)  = ATG ACG TCA ASA at posn = 36 2. VLS.fwdarw.VTS (L76T)  = GTG ACG TCA ASA at posn = 21 3. LAS.fwdarw.LTS (A107T) = TTG ACG TCA ASA at posn = 37 4. VTR.fwdarw.VTS (R138S) = GTG ACG TCA ASA at posn = 57 5. VLS.fwdarw.VTS (L167T) = GTG ACG TCA ASA at posn = 99

[0348] Inserting the CCAAT Binding Site into the DHFR Gene

[0349] CCAAT is usually defined as ATTGCGCAAT (SEQ ID NO: 9). Mutations in the DHFR gene can be made by inspection of the desired consensus sequence and all three frames and the corresponding changes to the amino acid sequence upon making the necessary single base-pair changes. The CCAAT is 10 bp and so can span five codons. For example, the sequence defined above can be put into any one of the three reading frames and the corresponding amino acid sequence and tolerated variations can be given:

TABLE-US-00017 A:Frame 1: ATT GCG CAA Txx 1:1 2:A 3:Q 4:FLSYCW B:Frame 2: xAT TGC GCA ATx 1:YHND 2:C 3:A 4:IM C:Frame 3: xxA TTG CGC AAT 1:LIVSPTAQKERG* 2:L 3:R 4:N

[0350] From this it is possible to implement changes into the mDHFR gene to give minimal perturbation to the overall sequence. Mutations should be placed at solvent exposed sites and away from the catalytic centre and where possible mutations should silent or conservative.

[0351] An example of an mDHFR gene that is modified to contain CCAAT is shown as follows:

TABLE-US-00018 ATGGTTCGACCATTGAACTGCATCGTCGCCGTGTCCCAAAATATGGGGATTGGCAAGAACGGAGACCTAC CCTGGCCTCCATTGCGCAATGAGTTCAAGTACTTCCAAAGAATGACCACAACCTCTTCAGTGGAAGGTAA ACAGAATCTGGTGATTATGGGTAGGAAAACCTGGTTCTCCATTCCTGAGAAGAATCGACCATTGCGCAAT AGAATTAATATAGTTCTCAGTAGAGAATTGCGCAATCCACCACGAGGAGCTCATTTTATTGCGCAATCCT TGGATGATGCATTGCGCAATATTGAACAACCGGAATTGGCGAGCAAAGTAGACATGGTTTGGATCGTCGG AGGCAGTTCTGTTTACCAGGAAGCCATGAATCAACCAGGCCACCTTAGACTCTTTGTGACAAGGATCATG CAGGAATTTGAAAGTGACACGTTTTTCCCAGAAATTGATTTGGGGAAATATAAACTTCTCCCAGAATACC CAGGCGTCCTCTCTGAATTGCGCAATGAAAAAGGCATCAAGTATAAGTTTGAAGTCTACGAGAAGAAAGA CTAAGCTTAA

[0352] Nucleotide residues in bold underline indicate CCAAT consensus binding sites.

[0353] Nucleotide residues in lowercase and italics correspond to the restriction enzyme sites for AscI and HindIII at the 5′ and 3′ ends of the sequence, respectively.

[0354] The resulting amino acid sequence is shows as follows:

TABLE-US-00019 M V R P L N C I V A V S Q N M G I G K N G D L P W P P L R N E F K Y F Q R M T T T S S V E G K Q N L V I M G R K T W F S I P E K N R P L   R I N I V L S R E L  P P R G A H F   A   S L D D A L R  I E Q P E L A S K V D M V W I V G G S S V Y Q E A M N Q P G H L R L F V T R I M Q E F E S D T F F P E I D L G K Y K L L P E Y P G V L S E V Q E E K G I K Y K F E V Y E K K D

[0355] Amino acid residues in italics are solvent exposed residues. The other residues are classed as buried residues.

[0356] Amino acid residues in bold underline are residues that have been altered as a result of the insertion of CCAAT into the nucleotide sequence.

[0357] A summary of the amino acid changes is provided as follows:

TABLE-US-00020 1. PLRN (silent) 2. PLKD.fwdarw.PLRN (K69R, D70N) = ASA at posns = 90, 97 3. ELKE.fwdarw.ELRN (K81R, E82N) = ASA at posns = 175, 131 4. LAKS.fwdarw.IAQS (L90I, K92Q) = ASA at posns = 47, 139 5. ALRL.fwdarw.ALRN (L100N)      = ASA at posn  =  46

[0358] A further CCAAT site could be inserted to make the following mutation:

TABLE-US-00021 6. EVQE.fwdarw.ELRN (V170L, Q171R, E172N)

[0359] Inserting the Ebox Binding Site into the DHFR Gene

[0360] In the context of cMyc, Ebox is usually defined as CACGTG (SEQ ID NO: 7) or CACATG (SEQ ID NO: 8). Mutations in the DHFR gene can be made by inspection of the desired consensus sequence and all three frames and the corresponding changes to the amino acid sequence upon making the necessary single base-pair changes. For example, the sequences defined above can be put into any one of the three reading frames and the corresponding amino acid sequence and tolerated variations can be given:

TABLE-US-00022 A:Frame 1: CAC GTG XXX = 1:H 2:V 3:Anything B:Frame 2: xCA CGT Gxx = 1:S/P/T/A 2:R 3:V/A/D/E/G C:Frame 3: xxC ACG TGx = 1:FLIVSPTAYHNDCRSG 2:T 3:C/W/* A:Frame 1: CAC ATG xxx = 1:H 2:M 3:anything B:Frame 2: xCA CAT Gxx = 1:S/P/T/A 2:H 3:V/A/D/E/G C:Frame 3: xxC ACA TGx = 1:FLIVSPTAYHNDCRSG 2:T 3:C/W/*

[0361] From this it is possible to implement changes into the mDHFR gene to give minimal perturbation to the overall sequence. Mutations should be placed at solvent exposed sites and away from the catalytic centre and where possible mutations should silent or conservative.

[0362] An example of an mDHFR gene that is modified to contain Eboxes is shown as follows:

TABLE-US-00023 ATGGTTCGACCATTGAACTGCATCGTCGCCGTGTCCCAAAATATGGGGATTGGCAAGAACGGAGACCTAC CCTGGCCTCCGCTCAGGAACGAGTTCAAGTACTTCCAAAGAATGACCACAACCTCTTCAGTGGAAGGTAA ACAGAATCTGGTGATTATGGGTAGGCGCACGTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGAC AGAATTAATATAGTTCTCTCACGTGAACTCAAAGAACCACCACGTGGAGCTCACGTGCTTGCCAAATCAC TGGATGATGCATTAAGACTTATTGAACAACCGGAATTGGCGTCACGTGTAGACATGGTTTGGATCGTCGG AGGCAGTTCTGTTTACCAGGAAGCCATGAATCAACCAGGCCACGTGAGACTCTTTGTGACACGTGTCATG CAGGAATTTGAAAGTGACACGTTTTTCCCAGAAATTGATTTGGGGAAATATAAACTTCTCCCAGAATACC CAGGCGTCCTCTCACGTGTCCAGGAGGAAAAAGGCATCAAGTATAAGTTTGAAGTCTACGAGAAGAAAGA CTAAGCTTAA

[0363] Nucleotide residues in bold underline indicate Ebox consensus binding sites.

[0364] Nucleotide residues in lowercase and italics correspond to the restriction enzyme sites for AscI and HindIII at the 5′ and 3′ ends of the sequence, respectively.

[0365] The resulting amino acid sequence is shows as follows:

TABLE-US-00024 M V R P L N C I V A V S Q N M G I G K N G D L P W P P L R N E F K Y F Q R M T T T S S V E G K Q N L V I M G R   T W F S I P E K N R P L K D R I N I V L S R E L K E P P R G A H   L A K S L D D A L R L I E Q P E L A S   V D M V W I V G G S S V Y Q E A M N Q P G H   R L F V T R   M Q E F E S D T F F P E I D L G K Y K L L P E Y P G V L S   V Q E E K G I K Y K F E V Y E K K D

[0366] Amino acid residues in italics are solvent exposed residues. The other residues are classed as buried residues.

[0367] Amino acid residues in bold underline are residues that have been altered as a result of the insertion of Ebox into the nucleotide sequence.

[0368] A summary of the amino acid changes is provided as follows:

TABLE-US-00025 1. KTW.fwdarw.RTW (K56R)  = CGC ACG TGG ASA at posn = 143 (exposed) 2. SRE   (silent)  = TCA CGT GAA ASA at posn = N/A 3. PRG   (silent)  = CCA CGT GGA ASA at posn = N/A 4. HFL.fwdarw.HVL (F89V)  = CAC GTG CTT ASA at posn = 71 (exposed) 5. SKV.fwdarw.SRV (K109R) = ACA CGT GTA ASA at posn = 109 (exposed) 6. HLR.fwdarw.HVR (L132V) = CAC GTG AGA ASA at posn = 1.6 7. TRI.fwdarw.TRV (1139V) = ACA CGT GTC ASA at posn =1.6 8. SEV.fwdarw.SRV (E151R) = ACA CGT GTC ASA at posn = 141 (exposed)

[0369] Changes 6 and 7 are located at residues that are classed as buried. Accordingly, constructs could be made that contain all 8 Ebox sites, one that is lacking site ‘6’, one that is lacking site ‘7’ and one that is lacking both sites ‘6’ and ‘7’ in order to determine whether the mutation at these ‘buried’ sites affect the function of the resultant DHFR protein.

REFERENCES

[0370] A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein.

REFERENCES

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[0415] For standard molecular biology techniques, see Sambrook, J., Russel, D. W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press

[0416] Sequence Annex

TABLE-US-00026 Amino acid sequence of wild-type murine dihydrofolate reductase (SEQ ID NO: 1) MVRPLNCIVAVSQNMGIGKNGDLPWPPLRNEFKYFQRMTTTSSVEGKQNLVIMGRKTWFSIPEKNRPLKD RINIVLSRELKEPPRGAHFLAKSLDDALRLIEQPELASKVDMVWIVGGSSVYQEAMNQPGHLRLFVTRIM QEFESDTFFPEIDLGKYKLLPEYPGVLSEVQEEKGIKYKFEVYEKKD Amino acid sequence of engineered murine dihydrofolate reductase (SEQ ID NO: 2) MVRPLNCIVAVSQNMGIGKNGDLPWPPLRNESKYFQRMTQTDSVESKQNLVIMGRKTWFSIPESNRPLKD RINIVLSQELKEPPRGAHFLAKSLDDALRLIESPELASKVDSVWIVGGSSVYQEAMTQPGHLRLFVTQIM QEFESDTFFPEIDSGKYKLLPESPGVLSQVQEEKGIKYKFEVYEKKD Amino acid sequence of wild-type human dihydrofolate reductase (SEQ ID NO: 3) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKG RINLVLSRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVWIVGGSSVYKEAMNHPGHLKLFVTRIM QDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND Nucleic acid sequence for the protein coding sequence of engineered murine dihydrofolate reductase (SEQ ID NO: 4) ATGGTTCGACCATTGAACTGCATCGTCGCCGTGAGTCAGAATATGGGGATTGGCAAGAACGGAGACCTACCCTGGCC TCCGCTCAGGAATGAGTCAAAGTACTTCCAAAGAATGACTCAGACTGACTCAGTTGAGTCAAAACAGAATCTGGTGA TTATGGGTAGGAAAACCTGGTTCTCCATTCCTGAGTCAAATCGACCTTTAAAGGACAGAATTAATATAGTTCTGAGT CAAGAACTCAAAGAACCACCACGAGGAGCTCATTTTCTTGCCAAAAGTTTGGATGATGCCTTAAGACTTATTGAGTC ACCGGAATTGGCGAGCAAAGTTGACTCAGTTTGGATCGTCGGAGGCAGTTCTGTTTACCAGGAAGCCATGACTCAAC CAGGCCACCTTAGACTCTTTGTGACTCAGATCATGCAGGAATTTGAGTCAGACACGTTTTTCCCAGAAATTGACTCA GGGAAATATAAACTTCTCCCTGAGTCACCAGGCGTCCTGAGTCAGGTCCAGGAGGAAAAAGGCATCAAGTATAAGTT TGAAGTCTACGAGAAGAAAGACTAA Nucleic acid seguences of TPA response elements (TRE) tgactca (SEQ ID NO: 5) tgagtca (SEQ ID NO: 6) Nucleic acid seguences of Ebox response elements cacgtg (SEQ ID NO: 7) CACATG (SEQ ID NO: 8) Nucleic acid seguence of C/EBP protein response element ATTGCGCAAT (SEQ ID NO: 9) Nucleic acid seguence of cAMP response element (CRE) TGACGTCA (SEQ ID NO: 10) Nucleic acid seguences of Maf recognition elements (MAREs) TGCTGA.sup.G/.sub.CTCAGCA (SEQ ID NO: 32) tgctga.sup.GC/.sub.CGTCAGCA (SEQ ID NO: 33) Nucleic acid seauence of Par/CREB-2/PAP binding site TTACGTAA(SEQ ID NO: 34) Nucleic acid seauence of polynucleotide encodina enaineered murine dihvdrofolate reductase including restriction enzyme sites (SEQ ID NO: 11) GCTAGCGTTCGACCATTGAACTGCATCGTCGCCGTGAGTCAGAATATGGGGATTGGCAAGAACGGAGACCTACCCTG GCCTCCGCTCAGGAATGAGTCAAAGTACTTCCAAAGAATGACTCAGACTGACTCAGTTGAGTCAAAACAGAATCTGG TGATTATGGGTAGGAAAACCTGGTTCTCCATTCCTGAGTCAAATCGACCTTTAAAGGACAGAATTAATATAGTTCTG AGTCAAGAACTCAAAGAACCACCACGAGGAGCTCATTTTCTTGCCAAAAGTTTGGATGATGCCTTAAGACTTATTGA GTCACCGGAATTGGCGAGCAAAGTTGACTCAGTTTGGATCGTCGGAGGCAGTTCTGTTTACCAGGAAGCCATGACTC AACCAGGCCACCTTAGACTCTTTGTGACTCAGATCATGCAGGAATTTGAGTCAGACACGTTTTTCCCAGAAATTGAC TCAGGGAAATATAAACTTCTCCCTGAGTCACCAGGCGTCCTGAGTCAGGTCCAGGAGGAAAAAGGCATCAAGTATAA GTTTGAAGTCTACGAGAAGAAAGACTAA Nucleic acid sequences of example reading frames Example reading frame 1: tga ctc Axx (SEQ ID NO: 12) Example reading frame 2: xTG act cax (SEQ ID NO: 13) Example reading frame 3: xxT gac tca (SEQ ID NO: 14) Amino acid sequence of example reading frame 3 (SEQ ID NO: 15) FSYCLPHRITNVADG Amino acid seguence of example codon triplets containing TREs GTGAGTCAG (SEQ ID NO: 16) AATGAGTCA (SEQ ID NO: 17) ATGACTCAG (SEQ ID NO: 18) ACTGACTCA (SEQ ID NO: 19) GTTGAGTCA (SEQ ID NO: 20) CCTGAGTCA (SEQ ID NO: 21) CTGAGTCAA (SEQ ID NO: 22) ATTGAGTCA (SEQ ID NO: 23) GTTGACTCA (SEQ ID NO: 24) ATGACTCAA (SEQ ID NO: 25) GTGACTCAG (SEQ ID NO: 26) TTTGAGTCA (SEQ ID NO: 27) ATTGACTCA (SEQ ID NO: 28) CCTGAGTCA (SEQ ID NO: 29) CTGAGTCAG (SEQ ID NO: 30) Amino acid seguence of engineered murine dihvdrofolate reductase used during design process (SEQ ID NO: 31) * = stop codon ASVRPLNCIVAVSQNMGIGKNGDLPWPPLRNESKYFQRMTQTDSVESKQNLVIMGRKTWFSIPESNRPLK DRINIVLSQELKEPPRGAHFLAKSLDDALRLIESPELASKVDSVWIVGGSSVYQEAMTQPGHLRLFVTQI MQEFESDTFFPEIDSGKYKLLPESPGVLSQVQEEKGIKYKFEVYEKKD*A* Nucleotide sequence of an exemplary murine dihydrofolate reductase gene engineered to include CRE binding sites (SEQ ID NO: 36) ATGGTTCGACCATTGAACTGCATCGTCGCCGTGTCCCAAAATATGGGGATTGGCAAGAACGGAGACCTAC CCTGGCCTCCGCTCAGGAACGAGTTCAAGTACTTCCAAAGAATGACGTCAACCTCTTCAGTGGAAGGTAA ACAGAATCTGGTGATTATGGGTAGGAAAACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGAC AGAATTAATATAGTGACGTCAAGAGAACTCAAAGAACCACCACGAGGAGCTCATTTTCTTGCCAAAAGTT TGGATGATGCCTTAAGACTTATTGAACAACCGGAATTGACGTCAAAAGTAGACATGGTTTGGATCGTCGG AGGCAGTTCTGTTTACCAGGAAGCCATGAATCAACCAGGCCACCTTAGACTCTTTGTGACGTCAATCATG CAGGAATTTGAAAGTGACACGTTTTTCCCAGAAATTGATTTGGGGAAATATAAACTTCTCCCAGAATACC CAGGCGTGACGTCAGAGGTCCAGGAGGAAAAAGGCATCAAGTATAAGTTTGAAGTCTACGAGAAGAAAGA CTAAGCTTAA Amino acid secuence of an exemplary murine dihydrofolate reductase engineered to include CRE binding sites (SEQ ID NO: 37) MVRPLNCIVAVSQNMGIGKNGDLPWPPLRNEFKYFQRMTSTSSVEGKQNLVIMGRKTWFSIPEKNRPLKDRINIVTS RELKEPPRGAHFLAKSLDDALRLIEQPELTSKVDMVWIVGGSSVYQEAMNQPGHLRLFVTSIMQEFESDTFFPEIDL GKYKLLPEYPGVTSEVQEEKGIKYKFEVYEKKD Nucleotide seguence of an exemplary murine dihydrofolate reductase gene engineered to include CCAATbinding sites (SEQ ID NO: 38) ATGGTTCGACCATTGAACTGCATCGTCGCCGTGTCCCAAAATATGGGGATTGGCAAGAACGGAGACCTACCCTGGCC TCCATTGCGCAATGAGTTCAAGTACTTCCAAAGAATGACCACAACCTCTTCAGTGGAAGGTAAACAGAATCTGGTGA TTATGGGTAGGAAAACCTGGTTCTCCATTCCTGAGAAGAATCGACCATTGCGCAATAGAATTAATATAGTTCTCAGT AGAGAATTGCGCAATCCACCACGAGGAGCTCATTTTATTGCGCAATCCTTGGATGATGCATTGCGCAATATTGAACA ACCGGAATTGGCGAGCAAAGTAGACATGGTTTGGATCGTCGGAGGCAGTTCTGTTTACCAGGAAGCCATGAATCAAC CAGGCCACCTTAGACTCTTTGTGACAAGGATCATGCAGGAATTTGAAAGTGACACGTTTTTCCCAGAAATTGATTTG GGGAAATATAAACTTCTCCCAGAATACCCAGGCGTCCTCTCTGAATTGCGCAATGAAAAAGGCATCAAGTATAAGTT TGAAGTCTACGAGAAGAAAGAC TAAGCTTAA Amino acid secuence of an exemplary murine dihydrofolate reductase engineered to include CCAAT binding sites (SEQ ID NO: 39) MVRPLNCIVAVSQNMGIGKNGDLPWPPLRNEFKYFQRMTTTSSVEGKQNLVIMGRKTWFSIPEKNRPLRNRINIVLS RELRNPPRGAHFIAQSLDDALRNIEQPELASKVDMVWIVGGSSVYQEAMNQPGHLRLFVTRIMQEFESDTFFPEIDL GKYKLLPEYPGVLSEVQEEKGIKYKFEVYEKKD Nucleotide seguence of an exemplary murine dihydrofolate reductase gene engineered to include Eboxes (SEQ ID NO: 40) ATGGTTCGACCATTGAACTGCATCGTCGCCGTGTCCCAAAATATGGGGATTGGCAAGAACGGAGACCTAC CCTGGCCTCCGCTCAGGAACGAGTTCAAGTACTTCCAAAGAATGACCACAACCTCTTCAGTGGAAGGTAA ACAGAATCTGGTGATTATGGGTAGGCGCACGTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGAC AGAATTAATATAGTTCTCTCACGTGAACTCAAAGAACCACCACGTGGAGCTCACGTGCTTGCCAAATCAC TGGATGATGCATTAAGACTTATTGAACAACCGGAATTGGCGTCACGTGTAGACATGGTTTGGATCGTCGG AGGCAGTTCTGTTTACCAGGAAGCCATGAATCAACCAGGCCACGTGAGACTCTTTGTGACACGTGTCATG CAGGAATTTGAAAGTGACACGTTTTTCCCAGAAATTGATTTGGGGAAATATAAACTTCTCCCAGAATACC CAGGCGTCCTCTCACGTGTCCAGGAGGAAAAAGGCATCAAGTATAAGTTTGAAGTCTACGAGAAGAAAGA CTAAGCTTAA Amino acid seauence of an exemolarv murine dihvdrofolate reductase enaineered to include Eboxes (SEQ ID NO: 41) MVRPLNCIVAVSQNMGIGKNGDLPWPPLRNEFKYFQRMTTTSSVEGKQNLVIMGRRTWFSIPEKNRPLKDRINIVLS RELKEPPRGAHVLAKSLDDALRLIEQPELASRVDMVWIVGGSSVYQEAMNQPGHVRLFVTRVMQEFESDTFFPEIDL GKYKLLPEYPGVLSRVQEEKGIKYKFEVYEKKD Nucleotide seauence of p300-mDHFR olasmid used in Examoles (SEQ ID NO: 42) CTCGAGAAATCATAAAAAATTTATTTGCTTTGTGAGCGGATAACAATTATAATAGATTCAATTGTGAGCGGATAACA ATTTCACACAGAATTCATTAAAGAGGAGAAATTAAGCATGCACCATCACCATCACCATgctagcgttcgaccattga actgcatcgtcgccgtgagtcagaatatggggattggcaagaacggagacctaccctggcctccgctcaggaatgag tcaaagtacttccaaagaatgactcagactgactcagttgagtcaaaacagaatctggtgattatgggtaggaaaac ctggttctccattcctgagtcaaatcgacctttaaaggacagaattaatatagttctgagtcaagaactcaaagaac caccacgaggagctcattttcttgccaaaagtttggatgatgccttaagacttattgagtcaccggaattggcgagc aaagttgactcagtttggatcgtcggaggcagttctgtttaccaggaagccatgactcaaccaggccaccttagact ctttgtgactcagatcatgcaggaatttgagtcagacacgtttttcccagaaattgactcagggaaatataaacttc tccctgagtcaccaggcgtcctgagtcaggtccaggaggaaaaaggcatcaagtataagtttgaagtctacgagaag aaagactaagcttAATTAGCTGAGCTTGGACTCCTGTTGATAGATCCAGTAATGACCTCAGAACTCCATCTGGATTT GTTCAGAACGCTCGGTTGCCGCCGGGCGTTTTTTATTGGTGAGAATCCAAGCTAGTTTGGGAGGTTCCAACTTTCAC CATAATGAAATAAGATCACTACCGGGCGTATTTTTTGAGTTATCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGA GAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAG TTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCAC AAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAGTTCCGTATGGCAATGAAAGA CGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGC TCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAAC CTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTT TGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACA AGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAA TTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATTTTTTTAAGGCAGTTATTGGTGCCCTTAAACGCCTGG GGTAATGACTCTCTAGCTTGAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGT TGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCCTCTAGAGCTGCCTCGCGCGTTTCGGTGATGACGGT GAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCG TCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATA CTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCG TAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGG CGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTG AGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTG ACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCC CCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGG AAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTG TGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACAC GACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTT GAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCG GAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAG ATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAA CTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTT TTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCA GCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACC ATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAG CCGCGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACC CAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAA AGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGT TATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCG AAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCT TTCGTCTTCAC Nucleotide sequence of p230d-basic-cJun plasmid used in Examples (SEQ ID NO: 43) CTCGAGAAATCATAAAAAATTTATTTGCTTTGTGAGCGGATAACAATTATAATAGATTCAATTGTGAGCGGATAACA ATTTCACACAGAATTCATTAAAGAGGAGAAATTAAGCATGCGCATTAAAGCCGAACGCAAACGGATGCGCAACCGCA TCGCAGCCTCCAAGTGCCGCAAACGCAAATTGGAGCGCATCGCCCGCTTGGAAGAAAAGGTGAAAACCCTGAAAGCA CAGAACTATGAGCTGGCCTCCACCGCCAACATGTTGCGCGAACAGGTGGCCCAGCTCGGCGCGCCTCATCACCATCA CCATCACTGATAAAGCGCGCCTTGATAAGCTTAATTAGCTGAGCTTGGACTCCTGTTGATAGATCCAGTAATGACCT CAGAACTCCATCTGGATTTGTTCAGAACGCTCGGTTGCCGCCGGGCGTTTTTTATTGGTGAGAATCCAGGCGAGATT TTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTA AAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTT TTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCA TCCGGAATTTCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCC ATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCG CAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGC CAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCA TGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGC TTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATTTTTTTAAGG CAGTTATTGGTGCCCTTAAACGCCTGGGGTAATGACTCTCTAGCTTGAGGCATCAAATAAAACGAAAGGCTCAGTCG AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCCTCTAGAGC TGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTA AGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCC AGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATA TGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGAC TCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATC AGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGG CGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACA GGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGG ATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGT AGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTAT CGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAG GTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCT GCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGC GGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTAC GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCT AGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAA TGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTA GATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTC CAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATC CAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGC TACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTA CATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCA GTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGAC TGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGG ATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGG ATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCAC CAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAA TACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAA TGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCAT TATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAC Nucleotide seauence ofoREP4 exoressina the lac repressor used in Examples (SEQ ID NO: 44) AAGCTTCACGCTGCCGCAAGCACTCAGGGCGCAAGGGCTGCTAAAGGAAGCGGAACACGTAGAAAGCCAGTCCGCAG AAACGGTGCTGACCCCGGATGAATGTCAGCTACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCA GGTAGCTTGCAGTGGGCTTACATGGCGATAGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAG CTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGATCTGATGG CGCAGGGGATCAAGATCTGATCAAGAGACAGGATGACGGTCGTTTCGCATGCTTGAACAAGATGGATTGCACGCAGG TTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCG TGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAG GACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGC GGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAG TATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAA CATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGG GCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCG ATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCG GACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCT CGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGG GACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCT ATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAG TTCTTCGCCCACCCCGGGCTCGATCCCCTCGCGAGTTGGTTCAGCTGCTGCCTGAGGCTGGACGACCTCGCGGAGTT CTACCGGCAGTGCAAATCCGTCGGCATCCAGGAAACCAGCAGCGGCTATCCGCGCATCCATGCCCCCGAACTGCAGG AGTGGGGAGGCACGATGGCCGCTTTGGTCGACAATTCGCGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCT TTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGG GCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGT TGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACA TGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGC GCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATG GTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATA TTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGAC CCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGG TCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGG ATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTC GTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGC GCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTT GGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCA CCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACC ACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCAACGTA AATGCATGCCGCTTCGCCTTCGCGCGCGAATTGTCGACCCTGTCCCTCCTGTTCAGCTACTGACGGGGTGGTGCGTA ACGGCAAAAGCACCGCCGGACATCAGCGCTAGCGGAGTGTATACTGGCTTACTATGTTGGCACTGATGAGGGTGTCA GTGAAGTGCTTCATGTGGCAGGAGAAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCG CTTCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGCGGAGAT TTCCTGGAAGATGCCAGGAAGATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCTCCGC CCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGC GTTTCCCCTGGCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCC GCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCC CCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACC ACTGGCAGCAGCCACTGGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGG ACAAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAA ACCGCCCTGCAAGGCGGTTTTTTCGTTTTCAGAGCAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATC TTATTAATCAGATAAAATATTTCTAGATTTCAGTGCAATTTATCTCTTCAAATGTAGCACCTGAAGTCAGCCCCATA CGATATAAGTTGTTAATTCTCATGTTTGACAGCTTATCATCGAT Nucleotide sequence of control cJun plasmid lacking the DNA-binding basic region used in Examples (SEQ ID NO: 45) CTCGAGAAATCATAAAAAATTTATTTGCTTTGTGAGCGGATAACAATTATAATAGATTCAATTGTGAGCGGATAACA ATTTCACACAGAATTCATTAAAGAGGAGAAATTAAGCATGCACCATCACCATCACCATGCTAGCATCGCCCGGCTGG AGGAAAAAGTGAAGACCTTGAAGGCCCAGAACTATGAGCTGGCGTCCACGGCCAACATGCTCCGGGAACAGGTGGCA CAGCTTGGCGCGCCTTAAGGTAGCTCTAAGCTTAATTAGCTGAGCTTGGACTCCTGTTGATAGATCCAGTAATGACC TCAGAACTCCATCTGGATTTGTTCAGAACGCTCGGTTGCCGCCGGGCGTTTTTTATTGGTGAGAATCCAGGCGAGAT TTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGT AAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTT TTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTT AATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATTTTTTTAAGGCAGTTATTGGTGCCCTTAAAC GCCTGGGGTAATGACTCTCTAGCTTGAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTT ATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCCTCTAGAGCTGCCTCGCGCGTTTCGGTGAT GACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACA AGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAG TGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACA GATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGG CTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAA CATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCC CCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCG TTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCC TTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGG GCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTA AGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGA GTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTA CCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAG CAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAA CGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAAT GAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCT ATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGG CTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACC AGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGG GAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACG CTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCA AAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATG GCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTC ATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCA GAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCC AGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAA AACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTC AATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAA ATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTA TAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAC Nucleotide sequence of basic-c-Jun (SEQ ID NO: 46) CGCATTAAAGCCGAACGCAAACGGATGCGCAACCGCATCGCAGCCTCCAAGTGCCGCAAACGCAAATTGGAGCGC Amino acid sequence of basic-c-Jun (SEQ ID NO: 47) RIKAERKRMRNRIAASKCRKRKLER Nucleotide sequence of Aβ.sub.1-42 (SEQ ID NO: 48) GACGCTGAATTTCGCCACGACTCCGGCTATGAGGTACACCACCAGAAACTGGTTTTTTTTGCTGAGGACGTTGGCTC CAACAAAGGTGCTATCATCGGTCTGATGGTTGGCGGCGTTGTTATCGCTTAA Amino acid sequence of Aβ.sub.1-42 (SEQ ID NO: 49) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA Nucleotide sequence of basic-Aβ.sub.1-42 (SEQ ID NO: 50) Sequence encoding Aβ.sub.1-42 underlined ATGCGCATTAAAGCCGAACGCAAACGGATGCGCAACCGCATCGCAGCCTCCAAGTGCCGCAAACGCAAATTGGAGCG CGACGCTGAATTTCGCCACGACTCCGGCTATGAGGTACACCACCAGAAACTGGTTTTTTTTGCTGAGGACGTTGGCT CCAACAAAGGTGCTATCATCGGTCTGATGGTTGGCGGCGTTGTTATCGCTTAA Amino acid sequence of basic-Aβ.sub.1-42 (SEQ ID NO: 51) Sequence encoding Aβ.sub.1-42 underlined MRIKAERKRMRNRIAASKCRKRKLERDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA Nucleotide sequence of αS (SEQ ID NO: 52) GATGTATTCATGAAAGGACTTTCAAAGGCCAAGGAGGGAGTTGTGGCTGCTGCTGAGAAAACCAAACAGGGTGTGGC AGAAGCAGCAGGAAAGACAAAAGAGGGTGTTCTCTATGTAGGCTCCAAAACCAAGGAGGGAGTGGTGCATGGTGTGG CAACAGTGGCTGAGAAGACCAAAGAGCAAGTGACAAATGTTGGAGGAGCAGTGGTGACGGGTGTGACAGCAGTAGCC CAGAAGACAGTGGAGGGAGCAGGGAGCATTGCAGCAGCCACTGGCTTTGTCAAAAAGGACCAGTTGGGCAAGAATGA AGAAGGAGCCCCACAGGAAGGAATTCTGGAAGATATGCCTGTGGATCCTGACAATGAGGCTTATGAAATGCCTTCTG AGGAAGGGTATCAAGACTACGAACCTGAAGCCTAA Amino acid sequence of αS (SEQ ID NO: 53) DVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVA QKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA Nucleotide sequence of basic-αS (SEQ ID NO: 54) Sequence encoding αS underlined ATGCGCATTAAAGCCGAACGCAAACGGATGCGCAACCGCATCGCAGCCTCCAAGTGCCGCAAACGCAAATTGGAGCG CGATGTGTTTATGAAAGGTCTGAGCAAAGCGAAAGAAGGCGTGGTGGCTGCGGCGGAAAAAACGAAACAGGGCGTGG CGGAAGCGGCCGGCAAAACGAAAGAAGGTGTTCTGTATGTCGGCAGCAAAACCAAAGAAGGCGTGGTTCATGGTGTG GCCACCGTTGCAGAAAAAACGAAAGAACAGGTCACCAACGTGGGCGGTGCTGTCGTGACCGGTGTTACGGCTGTCGC GCAAAAAACGGTGGAAGGCGCGGGTTCTATTGCGGCGGCAACCGGTTTCGTTAAAAAAGATCAGCTGGGTAAAAATG AAGAAGGCGCGCCGCAAGAAGGTATCCTGGAAGACATGCCGGTGGATCCGGACAACGAAGCGTATGAAATGCCGTCG GAAGAAGGCTATCAAGACTATGAACCGGAAGCGTAATGA Amino acid sequence of basic-αS (SEQ ID NO: 55) Sequence encoding αS underlined MRIKAERKRMRNRIAASKCRKRKLERDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTK EGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILED MPVDPDNEAYEMPSEEGYQDYEPEA Nucleic acid sequences of binding sites CAAT box GGCCAATCT (SEQ ID NO: 35) CArG box CC(A/T.sub.6)GG (SEQ ID NO: 56) E2 box CAGGTG and CACCTG (SEQ ID NOs: 57 and 58) HY box TG(A/T)GGG (SEQ ID NO: 59) T box TCACACCT (SEQ ID NO: 60) TATA box TATAAA (SEQ ID NO: 61) X box GTTGGCATGGCAAC (SEQ ID NO: 62) Y box (A/G)CTAACC(A/G)(A/G)(C/T) (SEQ ID NO: 63) ATA box AAATAT (SEQ ID NO: 64) CGCG box (A/C/G)CGCG(C/G/T) (SEQ ID NO: 65) DREB box TACCGACAT (SEQ ID NO: 66) Fur box GATAATGATAATCATTATC (SEQ ID NO: 67) G box GCCACGTGGC (SEQ ID NO: 68) GCC box AGCCGCC (SEQ ID NO: 69) H box ACACCA (SEQ ID NO: 70) Prolamin box TGTAAAG (SEQ ID NO: 71) Pyrimidine box CCTTTT (SEQ ID NO: 72) TACTAAC box ATTTACTAAC (SEQ ID NO: 73)

[0417] Numbered Clauses

[0418] The following numbered clauses, describing aspects and embodiments of the invention, are part of the description.

[0419] 1. A method for screening for an inhibitor of association between first and second candidate binding partners, the method comprising:

[0420] providing a cell, wherein the cell comprises:

[0421] a test compound;

[0422] a first hybrid protein comprising a first component of a DNA-binding protein linked to the first candidate binding partner;

[0423] a second hybrid protein comprising a second component of the DNA-binding protein linked to the second candidate binding partner; and

[0424] a reporter expression cassette that encodes a reporter expression product,

[0425] wherein the first and second hybrid proteins form a DNA-binding complex upon association of the first and second candidate binding partners, and wherein the reporter expression cassette comprises at least one binding site for the DNA-binding complex such that binding of the complex to the binding site inhibits expression of the reporter expression product; and

[0426] determining expression of the reporter expression product in the presence of the test compound;

[0427] wherein an increase in expression of the reporter expression product in the presence of the test compound indicates that the test compound is capable of inhibiting association between the first and second candidate binding partners.

[0428] 2. The method of clause 1, wherein the reporter expression product is a reporter protein.

[0429] 3. The method of clause 2, wherein the reporter protein is a cell survival protein, a cell reproduction protein a fluorescent protein, a bioluminescent protein, a protease, an enzyme that acts on a substrate to produce a colorimetric signal, a protein kinase, a transcriptional activator, or a regulatory protein such as ubiquitin.

[0430] 4. The method of clause 3, wherein the reporter protein is a cell survival protein, optionally wherein the cell survival protein is an enzyme involved in synthesising compounds that are required for cell survival, or a protein that is able to inhibit action of a toxic agent.

[0431] 5. The method of clause 3, wherein the reporter protein is a cell reproduction protein, optionally wherein the cell reproduction protein is an enzyme involved in synthesising compounds that are required for cell proliferation.

[0432] 6. The method of clause 4, wherein the cell survival protein is an exogenous cell survival protein that is able to compensate for a deficiency in an endogenous cell survival protein; and

[0433] wherein the method is performed under selection conditions such that survival of the cell is dependent upon activity of the exogenous cell survival protein.

[0434] 7. The method of clause 5, wherein the cell reproduction protein is an exogenous cell reproduction protein that is able to compensate for a deficiency in an endogenous cell reproduction protein; and

[0435] wherein the method is performed under selection conditions such that proliferation of the cell is dependent upon activity of the exogenous cell reproduction protein.

[0436] 8. The method of clause 6 or clause 7, wherein the exogenous cell survival protein is an orthologue of the endogenous cell survival protein, or the exogenous cell reproduction protein is an orthologue of the endogenous cell reproduction protein.

[0437] 9. The method of any one of clauses 6 to 8, wherein the exogenous cell survival protein or exogenous cell reproduction protein is resistant to selection conditions that inhibit the function of the endogenous cell survival protein or endogenous cell reproduction protein.

[0438] 10. The method of any one of clauses 6 to 9, wherein the selection conditions comprise the addition of a selection agent that inhibits the function of the endogenous cell survival protein or endogenous cell reproduction protein.

[0439] 11. The method of any one of clauses 4, 6, or 8 to 10, wherein the cell survival protein is dihydrofolate reductase (DHFR), optionally wherein the DHFR has an amino acid sequence that is at least 80% identical to the sequence set forth in SEQ ID NO: 1.

[0440] 12. The method of any one of the preceding clauses, wherein the reporter expression cassette comprises between 1 and 5, between 1 and 10, between 1 and 15, between 1 and 20, between 5 and 10, between 5 and 15, between 5 and 20, between 10 and 15, between 10 and 20, between 10 and 18 or between 12 and 16 binding sites.

[0441] 13. The method of any one of clauses 1 to 11, wherein the reporter expression cassette comprises at least 2, at least 5, at least 10, at least 12, or at least 15 binding sites.

[0442] 14. The method of any one of clauses 2 to 13, wherein the reporter protein retains at least 50%, at least 70%, at least 90%, or at least 95% of the function of a parent reporter protein, and wherein the parent reporter protein is encoded by a parent reporter expression cassette that corresponds to the reporter expression cassette but does not comprise the binding site(s).

[0443] 15. The method of any one of clauses 2 to 14, wherein some or all of the binding site(s) are located in the protein coding sequence of the reporter expression cassette.

[0444] 16. The method of clauses 15, wherein the majority or all of the binding sites located in the protein coding sequence of the reporter expression cassette were introduced as silent, semi-conservative and/or conservative mutations.

[0445] 17. The method of clause 15 or clause 16, wherein the majority or all of the binding sites located in the protein coding sequence of the reporter expression cassette are located at positions that encode a solvent exposed residue in the reporter protein.

[0446] 18. The method of any one of clauses 15 to 17, wherein the majority or all of the binding sites located in the protein coding sequence of the reporter expression cassette are not located at positions that encode a residue that forms part of the catalytic centre of the reporter protein.

[0447] 19. The method of any one of clauses 2 to 18, wherein the reporter protein has an amino acid sequence that is at least 80% identical to a parent reporter protein, wherein the parent reporter protein is encoded by a parent reporter expression cassette that corresponds to the reporter expression cassette but does not comprise the binding site(s).

[0448] 20. The method of any one the preceding clauses, wherein the method comprises administering the reporter expression cassette in order to provide the cell comprising the reporter expression cassette.

[0449] 21. The method of any one of the preceding clauses, wherein the first and second components of the DNA-binding protein have an identical amino acid sequence.

[0450] 22. The method of any one of the preceding clauses, wherein the first and second components of the DNA-binding protein have different amino acid sequences.

[0451] 23. The method of any one of the preceding clauses, wherein the first and second components of the DNA-binding protein lack a dimerization domain.

[0452] 24. The method of any one of the preceding clauses, wherein the first and second components of the DNA-binding protein are DNA-binding fragments of a transcription factor.

[0453] 25. The method of clause 23, wherein the transcription factor is a eukaryotic transcription factor, optionally a human transcription factor.

[0454] 26. The method of any one of the preceding clauses, wherein the DNA-binding complex lacks a functional domain for activating transcription of the reporter expression product.

[0455] 27. The method of any one of clauses 24 to 26, wherein the first and second components of the DNA-binding protein are DNA-binding fragments of a basic leucine zipper (bZIP), basic helix-loop helix (bHLH) or bHLH leucine zipper (bHLH-Zip) transcription factor, and optionally wherein [0456] a) the at least one binding site is a TPA response element (TRE) having the nucleotide sequence TGACTCA (SEQ ID NO: 5) or TGAGTCA (SEQ ID NO: 6); [0457] b) the at least one binding site is an Ebox response element having the nucleotide sequence CACGTG (SEQ ID NO: 7) or CACATG (SEQ ID NO: 8); [0458] c) the at least one binding site is a CCAAT binding site having the nucleotide sequence ATTGCGCAAT (SEQ ID NO: 9); [0459] d) the at least one binding site is a cAMP response element (CRE) having the nucleotide sequence TGACGTCA (SEQ ID NO: 10); or [0460] e) the at least one binding site is a Maf recognition element (MARE) having the nucleotide sequence TGCTGA.sup.G/.sub.CTCAGCA (SEQ ID NO: 32) or TGCTGA.sup.GC/.sub.CGTCAGCA (SEQ ID NO: 33); [0461] f) the at least one binding site is a PAP/CREB-2/PAR binding site having the nucleotide sequence TTACGTAA (SEQ ID NO: 34).

[0462] 28. The method of clause 27, wherein the transcription factor is a member of the Fos/Jun subfamily of transcription factors (such as c-Jun), optionally wherein the first and second components of the DNA-binding protein each comprise an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 47.

[0463] 29. The method of clause 28, wherein the reporter expression cassette comprises a nucleotide sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 4.

[0464] 30. The method of any one of the preceding clauses, wherein the first and second candidate binding partners have an identical amino acid sequence.

[0465] 31. The method of any one of clauses 1 to 29, wherein the first and second candidate binding partners have different amino acid sequences.

[0466] 32. The method of any one of the preceding claims, wherein the first and second candidate binding partners are capable of forming aggregates, optionally wherein the first and second candidate binding partners are capable of aggregating to form amyloids or amorphous deposits.

[0467] 33. The method of any one of the preceding clauses, wherein aggregation of the first and second candidate binding partners in a human patient is associated with a disease, optionally wherein the disease is a neurodegenerative disease.

[0468] 34. The method of any one of the preceding clauses, wherein the first and second candidate binding partners are amyloid peptides.

[0469] 35. The method of clause 34, wherein the first and second candidate binding partners are amyloid-β (Aβ) peptides, optionally wherein the Aβ peptides comprise an amino acid sequence having the sequence of SEQ ID NO: 49.

[0470] 36. The method of clause 35, wherein the first and second hybrid proteins each comprise an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 51.

[0471] 37. The method of clause 34, wherein the first and second candidate binding partners are prion proteins (PrPs).

[0472] 38. The method of clause 34, wherein the first and second candidate binding partners are tau proteins.

[0473] 39. The method of clause 34, wherein the first and second candidate binding partners are α-synuclein (αS) polypeptides, optionally wherein the αS polypeptides comprise an amino acid sequence having the sequence of SEQ ID NO: 53.

[0474] 40. The method of clause 39, wherein the first and second hybrid proteins each comprise an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 55.

[0475] 41. The method of any one of the preceding clauses, wherein the first hybrid protein is a first fusion protein comprising the first component of the DNA-binding protein and the first candidate binding partner in the same polypeptide chain, and wherein the second hybrid protein is a second fusion protein comprising the second component of the DNA-binding protein and the second candidate binding partner in the same polypeptide chain.

[0476] 42. The method of clause 41, wherein method comprises administering a fusion protein expression cassette that encodes both the first and second fusion proteins to the cell such that the cell expresses the first and second fusion proteins.

[0477] 43. The method of clause 42, wherein the fusion protein expression cassette comprises a nucleotide sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 50.

[0478] 44. The method of clause 42, wherein the fusion protein expression cassette comprises a nucleotide sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 54.

[0479] 45. The method of any one of the preceding clauses, wherein the first and second hybrid proteins have an identical amino acid sequence.

[0480] 46. The method of any one of the preceding clauses, wherein the cell is a bacterial cell, optionally an Escherichia coli cell.

[0481] 47. The method of any one of clauses 1 to 45, wherein the cell is a eukaryotic cell.

[0482] 48. The method of clause 47, wherein the eukaryotic cell is a mammalian cell.

[0483] 49. The method of any one of the preceding clauses, wherein the test compound is a peptidic compound or a small molecule.

[0484] 50. The method of clause 51, wherein the test compound is a peptidic compound.

[0485] 51. The method of clause 50, wherein the compound is expressed intracellularly from a test compound expression cassette.

[0486] 52. The method of clause 51, wherein the method comprises providing the test compound expression cassette to the cell.

[0487] 53. The method of any one of clauses 50 to 52, wherein the method comprises administering a cross-linking agent into the cell in order to introduce a cross-link between two amino acid residues in an alpha helix of the peptidic test compound to produce a helix-constrained peptidic compound.

[0488] 54. The method of clause 53, wherein the method comprises determining expression of the reporter expression product both before and after the addition of the cross-linking agent.

[0489] 55. The method of clause 49 or clause 50, wherein the method comprises administering the test compound extracellularly in order to provide the cell comprising the test compound, optionally wherein an increase in expression of the reporter expression product indicates that the test compound is capable of entering the cell as well as being capable of inhibiting association between the first and second candidate binding partners.

[0490] 56. The method of clause 55, wherein the test compound is a peptidic test compound, wherein the peptidic test compound comprises a helix-constrained peptide, and wherein the helix-constrained peptide comprises a cross-link between two amino acid residues.

[0491] 57. The method of clause 53 or clause 56, wherein the cross-link is formed between residues i and i+4 in the peptidic test compound.

[0492] 58. The method of any one of clauses 53, clause 56 or 57, wherein the cross-link is formed between cysteine residues in the peptidic test compound.

[0493] 59. A method for screening for an inhibitor of association between first and second candidate binding partners, the method comprising:

[0494] providing a cell free expression system comprising:

[0495] a test compound;

[0496] a first hybrid protein comprising a first component of a DNA-binding protein linked to the first candidate binding partner;

[0497] a second hybrid protein comprising a second component of the DNA-binding protein linked to the second candidate binding partner; and

[0498] a reporter expression cassette that encodes a reporter expression product,

[0499] wherein the first and second hybrid proteins form a DNA-binding complex upon association of the first and second candidate binding partners, and wherein the reporter expression cassette comprises at least one binding site for the DNA-binding complex such that binding of the DNA-binding complex to the binding site inhibits expression of the reporter expression product; and

[0500] determining expression of the reporter expression product;

[0501] wherein an increase in expression of the reporter expression product in the presence of the test compound indicates that the test compound is capable of inhibiting association between the first and second candidate binding partners.

[0502] 60. The method of any one of the preceding clauses, wherein the method further comprising carrying out an in vitro assay to confirm binding of the test compound to the first and/or second candidate binding partners.

[0503] 61. The method of clause 60, wherein the in vitro assay comprises carrying out one or more of surface plasmon resonance (SPR), isothermal calorimetry and X-ray crystallography.

[0504] 62. A fusion protein comprising a fusion protein comprising a component of a DNA-binding protein and a component of a DNA-binding protein and an amyloid peptide component capable of dimerization;

[0505] wherein said fusion protein forms a complex capable of binding DNA upon dimerization via the amyloid peptide component.

[0506] 63. A fusion protein according to clause 62 wherein the amyloid peptide component is an amyloid-β (Aβ) peptide or an α-synuclein (αS) polypeptide.

[0507] 64. The fusion protein of clause 63, wherein the Aβ peptide has the amino acid sequence set forth in SEQ ID NO: 49.

[0508] 65. The fusion protein of clause 64, wherein the αS polypeptide has the amino acid sequence set forth in SEQ ID NO: 53.

[0509] 66. The fusion protein of any one of clauses 63 to 65, wherein the DNA-binding component is a DNA-binding fragment of a member of the Fos/Jun subfamily of transcription factors (such as c-Jun).

[0510] 67. The fusion protein of clause 66, wherein the DNA-binding component comprises an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 47.

[0511] 68. The fusion protein of clause 66 or clause 67, wherein the fusion protein comprises an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 51.

[0512] 69. The fusion protein of clause 66 or clause 67, wherein the fusion protein comprises an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 55.

[0513] 70. A fusion protein expression cassette encoding the fusion protein of any one of clauses 62 to 69. 71. A kit comprising:

[0514] a reporter expression cassette that encodes a reporter expression product; and

[0515] one or more fusion protein expression cassettes encoding a first and second fusion protein;

[0516] wherein the first fusion protein comprises a first component of a DNA-binding protein and a first candidate binding partner,

[0517] wherein the second fusion protein comprises a second component of a DNA-binding protein and a second candidate binding partner,

[0518] wherein the first and second fusion proteins form a DNA-binding complex upon association of the first and second candidate binding partners; and

[0519] wherein the reporter expression cassette comprises at least one binding site for the DNA-binding complex such that binding of the DNA-binding complex to the binding site inhibits expression of the expression product.

[0520] 72. The kit of clause 71, wherein the first and second fusion proteins have an identical amino acid sequence and are both encoded by the same fusion protein expression cassette.

[0521] 73. The kit of clause 71, wherein the first and second fusion proteins have non-identical amino acid sequences, wherein the kit comprises a first fusion protein expression cassette encoding the first fusion protein and a second fusion expression cassette encoding the second fusion protein.

[0522] 74. The kit of any one of clauses 71 to 73, wherein the kit further comprises a test compound.

[0523] 75. A cell comprising:

[0524] a reporter expression cassette that encodes a reporter expression product; and

[0525] one or more fusion protein expression cassettes encoding a first and second fusion protein;

[0526] wherein the first fusion protein comprises a first component of a DNA-binding protein and a first candidate binding partner,

[0527] wherein the second fusion protein comprises a second component of a DNA-binding protein and a second candidate binding partner;

[0528] wherein the first and second fusion proteins form a DNA-binding complex upon association of the first and second candidate binding partners; and

[0529] wherein the reporter expression cassette comprises at least one binding site for the DNA-binding complex such that binding of the DNA-binding complex to the binding site inhibits expression of the expression product.